The application of the continuous density gradient cell separation method has been resumed and applied to isolate hematopoietic progenitor cells from human peripheral blood to compare the results to those obtained by the conventional density gradient centrifugation method. Recently, a new field of research called regenerative medicine has been established. Stem cells or progenitor cells are used for the restoration of injured or diseased tissues. Therefore, the selection criteria of the preparative cell separation methods should be focused on a multitude of target cells and satisfactory viability of separated cells. Our continuous-flow cell separation method with a Percoll density gradient originally developed for apheresis can separate a large number of cells into five fractions according to their densities. Nucleated cells (>10 million cells) present among a large population of red blood cells (>10 Billion cells) were separated from about 10 ml of human peripheral blood in about 100 minutes. We previously reported that lymphocytes were well separated from granulocytes, and progenitor cells were neither found in fraction 2 (density = 1.060 g/ml) nor fraction 6 (density = 1.080 g/ml). In fractions 3 to 5 a variety of progenitor cells such as burst-forming unit-erythroid (BFU-E), colony-forming unit-granulocyte, macrophage (CFU-GM) and colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM) were harvested with a minimum loss of their viability. Generally, low-density mononuclear cells such as monocytes, lymphocytes and progenitor cells are separated by a conventional density gradient centrifugation method with a Ficoll solution by adjusting the density at 1.077g/ml (Ficoll-Hypaque). In order to demonstrate the performance of our continuous-flow cell separation method, human peripheral blood from healthy adults was separated by the above two methods for comparison. In the conventional density gradient centrifugation method, progenitor cells were not only found at the plasma-Ficoll interface, but also in the Ficoll layer which is usually discarded. The progenitor cell frequencies determined by the colony forming-cell assay were 30147 colonies/ml blood separated by the conventional density gradient centrifugation method, whereas they were 56170 colonies/ml of blood separated by our continuous-flow cell separation method. These results suggest that the conventional density gradient centrifugation method would lose a considerable amount of progenitor cells and might induce cell injury, whereas our continuous-flow cell separation method could give a high yield of progenitor cells with minimal cell injury. Also, in the case of the separation of normal human peripheral blood by our method under a slightly hypertonic condition of 320 mOsmol/l, lymphocytes were shifted one layer toward the periphery and enriched in fractions 4 and 5 leaving progenitor cells behind. This result indicates that our method is able to sensitively detect a minute change in the cell densities produced under the hypertonic condition. At present, we are planning to manufacture the sterilized separation disk equipped with a circular channel and twelve tubes for disposable uses. The above results have been presented at the 5th International Conference on Countercurrent Chromatography which was held in Rio de Janeiro in July 26-30, 2008.